REL 2019-04-12 2019-09-30 2019-09-30 0000-0003-4738-9503 Max Wilkinson MRC Laboratory of Molecular Biology; University of Cambridge Francis Crick Avenue Cambridge Cambridgeshire United Kingdom CB2 0QH 0000-0003-1785-6510 Kiyoshi Nagai MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Cambridgeshire United Kingdom CB2 0QH Charenton C Wilkinson ME Nagai K 2.3 10.6019/EMPIAR-10307 EMDB EMD-4658 EMD-4673 EMD-4672 EMD-4674 EMD-4675 EMD-4676 EMD-4686 EMD-4665 EMD-4687 EMD-4688 EMD-4689 EMD-4690 Charenton C Wilkinson ME Nagai K Science (New York, N.Y.) Science 6438 364 362 367 2019 English 10.1126/science.aax3289 30975767

5 datasets containing collectively 28,705 multi-frame micrographs after motion correction and dose-weighting. Also contains particle coordinates, polished particles for the human tri-snRNP, unpolished particles for pre-B complex, and metadata star files from various refinements in Relion.

Dataset 1 of human pre-B spliceosome; motion-corrected micrographs /data/MotionCorr/job007 micrographs - single frame MRC MRC 4627 1 32 BIT FLOAT 3838 1.025 3710 1.025

Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 57 electrons per squared Angstrom over 8 seconds. Data collected on Titan Krios 2 at the Astbury Biostructure Laboratory, University of Leeds, on a K2 detector operated in counting mode. dataset1_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)

data/MotionCorr/job007/Raw_data/FoilHole_*_Data_*_*_201810*_*-*.mrc data/dataset1_CRYOLOpicking.star data Dataset 2 of human pre-B spliceosome; motion-corrected micrographs /data/MotionCorr/job593 micrographs - single frame MRC MRC 6869 1 32 BIT FLOAT 3838 1.022 3710 1.022

Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 50 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset2_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)

data/MotionCorr/job593/Movies_2Nov/FoilHole_*_Data_*_*_201811*_*-*.mrc data/dataset2_CRYOLOpicking.star data Dataset 3 of human pre-B spliceosome; motion-corrected micrographs /data/MotionCorr/job843/Movies_20Dec micrographs - single frame MRC MRC 7303 1 32 BIT FLOAT 3838 1.02 3710 1.02

Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 55.4 electrons per squared Angstrom over 12 seconds. Data collected on a Titan Krios at the University of Cambridge biochemistry department on a K2 detector operated in counting mode. dataset3_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)

data/MotionCorr/job843/Movies_20Dec/FoilHole_*_Data_*_*_201812*_*-*.mrc data/dataset3_CRYOLOpicking.star data Dataset 4 of human pre-B spliceosome; motion-corrected micrographs /data/MotionCorr/job890/Movies_11Jan micrographs - single frame MRC MRC 6672 1 32 BIT FLOAT 3838 1.022 3710 1.022

Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 48.6 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset4_CRYOLOpicking.star contains particle coordinates picked by CRYOLO (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)

data/MotionCorr/job890/Movies_11Jan/FoilHole_*_Data_*_*_201901*_*-*.mrc data/dataset4_CRYOLOpicking.star data Dataset 5 of human pre-B spliceosome; motion-corrected micrographs /data/MotionCorr/job1163/Movies_9Feb micrographs - single frame MRC MRC 3234 1 32 BIT FLOAT 3838 1.022 3710 1.022

Micrographs were averaged using RELION 3.0 implementation of MotionCor2 with 5x5 patches and dose-weighting. Original movies were 40 frames with a total dose of 44 electrons per squared Angstrom over 6 seconds. Data collected on Titan Krios 3 at the MRC Laboratory of Molecular Biology on a K2 detector operated in counting mode. dataset5_gautomatchpicking.star contains particle coordinates picked by Gautomatch using templates from pre-B complex 2D averages from the other 4 datasets (X and Y in 1st and 2nd columns; remainder of .star file is output of RELION particle extraction job, extracted particles not deposited here)

data/MotionCorr/job1163/Movies_9Feb/FoilHole_*_Data_*_*_201902*_*-*.mrc data/dataset5_gautomatchpicking.star data Selected U4/U6.U5 tri-snRNP particles after Bayesian polishing /data/Polish picked particles - single frame - processed MRCS MRCS 20537 585488 32 BIT FLOAT 420 1.022 420 1.022

Particles from datasets 2, 3, and 4 were picked with CRYOLO (see dataset*_CRYOLOpicking.star) and 3D classified. Tri-snRNP and pre-B particles were selected, CTF-refined and subjected to Bayesian polishing in RELION 3.0, giving the particle stacks in Polish/ A further round of CTF refinement produced the reconstruction deposited in the EMDB as EMD-4658, with the resultant run_data.star file deposited here as "trisnRNP_EMD4658_refine_data.star" Further classification of these particles yielded various other deposited maps, metadata for which are deposited here. In summary: "preB_unshiftedbox_EMD4665_refine_data.star" The subset of these polished tri-snRNP particles that correspond to pre-B complex, corresponds to EMD-4665 "trisnRNP_prp28class_EMD4686_refine_data.star" Subset of tri-snRNP with strong Prp28, corresponds to EMD-4686 "trisnRNP_U540Kclass_refine_data.star" Subset of tri-snRNP with strong U5-40K For full details see Charenton et al Science 2019.

Crude shifted preB particles /data/Extract picked particles - single frame - unprocessed MRCS MRCS 873597 1 32 BIT FLOAT 360 1.7033 360 1.7033

Particles from all datasets were picked (see dataset*_*picking.star) and 3D classified using a tri-snRNP reference. Tri-snRNP and pre-B particles were selected and re-extracted with a "shifted box" such that the box would encompass the full pre-B complex. Particles were also scaled down to 1.7033 Ångstrom per pixel. Further rounds of 3D classification produced a crude set of 117,284 pre-B particles, metadata in the deposited preB_allparticles_shiftedbox_refine_data.star Further classification of these particles yielded various deposited maps, metadata for which are deposited here. In summary: "preB_prp4kclass_EMD4688_refine_data.star" The subset of pre-B particles with strong density for Prp4 kinase, corresponds to EMD-4688 "preB_U1class_EMD4687_refine_data.star" The subset of pre-B particles with strong density for U1 snRNP, corresponds to EMD-4687 "preB_U2class_EMD4690_refine_data.star" The subset of pre-B particles with strong density for U2 snRNP, corresponds to EMD4690